This invention relates to a large animal model of human cancer, in particular in ruminant animals such as sheep which are immunosuppressed by cyclosporin A and ketoconazole and which carry transplanted human or murine tumours, or both. The invention also relates to the use of such an animal model in the study of cancer, particularly for evaluating candidates for radio-, chemo- or radiopharmaceutical therapy or radio-immunotherapy. The animal model is also useful for radio-imaging of neoplasms or tumours, and for the study of metastasis.
At present there is no effective method available for treatment of many solid tumours such as malignant melanoma or cancer of the colon, breast or ovary once the primary tumour has metastasised. Radiolabelled monoclonal antibodies against tumour-associated antigens offer a unique potential for targeting radiotherapy to disseminated tumour cells which may ultimately lead to effective treatment of metastatic cancer. Radioimmunotherapy has been shown to be effective in haematological malignancy, but problems of tumour localisation and penetration have so far prevented successful treatment of solid tumour metastasis.
In order to evaluate therapeutic agents, or methods of imaging tumours, and to study the biological processes taking place in the development and metastasis of solid tumours, it is essential to use animal models of cancer. The biodistribution of radiolabelled monoclonal antibodies can only be determined in the intact animal, where the influences of serum protein binding, vascular permeability, interstitial pressure and enzymatic breakdown all affect therapeutic radiation of the target tumour and determine the background irradiation of normal tissues. This essential dosimetry cannot be performed in vitro.
The immune-incompetent nude mouse, and less commonly, the nude rat, are the only models which are widely used for in vivo study of human tumours. The tumours are usually transplanted subcutaneously in these rodents. The major problem associated with human tumour xenografts in nude animals is the disproportionate size of the tumour in relation to the total body weight of the animals, which precludes accurate, predictive pharmacokinetic studies of potential chemotherapeutic and radiopharmaceutical treatments for human cancer, and adversely affects the usefulness of such models for imaging studies.
Similar problems are encountered in orthotopic implantation models, in which human tumours or tumour cells are transplanted or injected into the organ or tissue of origin in recipient immunodeficient athymic mice (Manzotti et al, 1993). Although metastasis of the transplanted tumour is achieved, accurate and reliable data on usefulness of therapeutic agents or methods are still limited by the disproportionate size of the tumour in relation to the total body weight of the mouse.
Therefore, a large animal model would be more suitable as a model of cancer and for detailed study of targeted cancer therapy. Large animal models of human cancer are not readily available, because of the difficulty of establishing tumours in such hosts; the xenografts usually do not grow or are rejected.
An animal model which would allow investigation of tumour nodules of a specific size and location, and which would simulate patterns of metastasis in various types of cancer, is particularly desirable. Larger animal models will also permit more effective and accurate evaluation of potential methods of therapy and imaging, and better characterisation of the biological events taking place during development and treatment of such cancers.
One way of inducing acceptance of xenografts is the administration of Cyclosporin A (CSA), a cyclic fungal peptide produced by Tolypocladium inflatum Gams. CsA is a neutral cycloundecapeptide with potent immunosuppressive properties (Borel, 1989; Di Padova, 1989; Hess et al, 1988). This antifungal metabolite appears to inhibit both humoral and cellular immune responses by selectively interfering with T-cell activation (Borel, 1989; Di Padova, 1989; Hess et al, 1988). CsA has been shown to be effective in preventing transplant rejection in both humans and animals, but its use is often limited by its toxic side-effects (Borel, 1989; Reynolds et al, 1992; Russ, 1992), and by the high concentrations required in order to induce immunosuppression. The normal vehicle used, Cremaphor EL, can also induce severe toxic side effects.
For example, rabbits given intramuscular injections of CsA at 10 mg/kg suffered from toxic side effects, and became anorexic and developed pneumonia. These effects were only eliminated if larger animals were used, and antibiotic and fluid therapy were instituted together with cyclosporin administration (Ligget et al, 1993). Cats also require high oral doses of CsA in order to accept human tumour xenografts, since intravenous administration is also associated with species-specific Cremaphor-induced vasoconstriction with histamine release and anaphylaxis (Bowers et al, 1991).
However, in sheep, infusion of the castor-oil based vehicle for CsA, Cremaphor EL, is well tolerated (Tresham et al, 1988). There is also no nephrotoxic reaction to intravenous CsA in sheep (Tresham et al, 1990). A recent pharmacokinetic study of CsA administered intravenously to sheep revealed data similar to that reported in human transplant patients (Charles et al, in press), and no toxic effects were described.
In addition to the toxic effects of CsA, a major disadvantage of this compound is the requirement for daily injections, which is both tedious and expensive and limits the period of time within which animals can be kept for observations (Hu et al, 1994, and de Ward-Siebinga et al, 1994). In all the studies mentioned above, the amount of CsA administered has been more than 10 mg/kg of animal weight.
There has been a single brief report of experiments in which human melanoma tumours have been subcutaneously grown in dogs immune-suppressed by oral CsA (Wiseman et al, 1991). This method, however, also requires high doses of CsA due to its limited bioavailability from oral administration. The intravenous route is precluded by the anaphylactic reaction of dogs to the Cremaphor vehicle in which cyclosporin is dissolved (Bowers et al, 1991).
More recently, several groups have reported the use of ketoconazole in conjunction with CsA as a means of reducing the dose of CsA required in transplant patients to maintain immunosuppression and prevent graft rejection (Gandhi et al, 1992; Butman et al, 1991; First et al, 1991; Wadhwa et al, 1987). Ketoconazole is a synthetic imidazole dioxolane used primarily for the treatment of superficial fungal infections, chronic mucocutaneous candidiasis and genital candidiasis (Bodey, 1992; Breckenridge, 1992; Borelli et al, 1979). Ketoconazole indirectly enhances the bioavailability of CsA by inhibiting the hepatic cytochrome P-450 mixed function oxidase system which is primarily responsible for CsA inactivation in vivo (Breckenridge, 1992; First et al, 1991; Wadhwa et al, 1987). Increased bioavailability reduces the dose of CsA required for therapeutic efficacy, which, in turn, decreases the toxicity associated with its use.
Ketoconazole, in addition to its synergism with CsA in the induction and maintenance of immunosuppression, has been reported to exert anti-tumour activity against certain types of cancer (Eichenberger et al, 1989a; Mahler and Denis, 1992). Ketoconazole also acts in synergy with anti-neoplastic drugs (vinblastine, etoposide) to inhibit the growth of human prostate carcinoma cells in vitro (Eichenberger et al, 1989b).
Similarly, CsA has been shown to inhibit cell division of both normal and malignant cells in vivo and in vitro (Borel, 1989; Di Padova, 1989; Barbera-Guillem et al, 1988; Kreis and Soricelli, 1979). Of the cell lines tested, human and murine T cell lymphomas and leukaemias were found to be sensitive to CsA-induced growth inhibition at doses of 0.5-5 xcexcg/ml, whereas non-lymphoid cell lines and certain murine B and null cell leukaemias were insensitive to doses of up to 10 xcexcg/ml (Borel, 1989).
We have surprisingly found that concomitant oral administration of ketoconazole and CsA to a mammal produces immunosuppression which allows xenografting of cancer cells or tissues and provides a large animal model for the study of cancer. This is particularly unexpected, in view of the anti-tumour effects of ketoconazole and CsA, and the difficulty of inducing and maintaining immunosuppression with non-toxic doses of CsA.
A main advantage of the animal model according to the present invention is the cost effectiveness of obtaining and maintaining the animals. No aseptic or sterile conditions are necessary and the animals can be maintained on a normal diet.
Our model also permits investigation of tumour nodules of desired size at predetermined sites, which simulate the usual patterns of metastasis of particular cancers.
Thus, in one aspect the invention provides an animal model of cancer, comprising a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole, and which carries a tumour xenograft.
Preferably, the mammal is selected from the group consisting of sheep, goats, cattle, pigs or the like. More preferably, the mammal is a sheep. In a particularly preferred embodiment, the mammal has a plurality of xenografted tumours implanted subcutaneously.
Tumour cell lines which may be used in this model include but are not limited to cells from solid tumours, such as those present in cancer of the colon, breast or ovary, or melanoma. Cell lines or spheroids derived from cancerous cells are particularly useful for the purposes of the invention, for example LS174T, HT-29 and colon cancer and SKMEL melanoma cell lines. The tumour may be of human or non-human origin, but is preferably of human or murine origin.
It is particularly preferred that the tumours are introduced into the model of the invention using orthotopic transplantation.
In a particularly preferred embodiment of the invention, tumour cells or tumours are transplanted into the host animal using Matrigel as the vehicle. Matrigel is a reconstituted basementxe2x80x94-membrane preparation which facilitates tumour uptake at sites of incubation.
In a second aspect, the invention provides a method of evaluating the efficacy of a putative therapeutic agent against cancer, comprising the step of administering said agent to a ruminant mammal model of the invention.
The agents which may be tested in this model include but are not limited to immunochemotherapeutic agents, cytokines, chemotherapeutic agents and radiopharmaceuticals, and may also comprise internal or external radioactive agents as well as radiolabelled peptides. Gene therapy may also be evaluated using this model.
In a third aspect, the invention provides a method of evaluating the efficacy of a method of radioimaging of tumours or neoplasms, comprising the step of administering a radiolabelled, tumour-specific antibody to the ruminant mammal model of the invention.
The radiolabelled antibody may be a monoclonal or polyclonal antibody comprising a radiolabel, preferably selected from the group consisting of Technetium-99m, Indium-111, Iodine-131, Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177, Copper-64, Scandium-47, Yttrium-90. Monoclonal antibodies labelled with therapeutic radionuclides such as Iodine-131, Rhenium-188, Holmium-166, Samarium-153 and Scandium-47, which do not compromise the immunoreactivity of antibodies and are not broken down in viva, are especially preferred. The person skilled in the art will appreciate that other radioactive isotopes are known, and may be suitable for specific applications. Similarly it will be clearly understood that the term xe2x80x9cantibodyxe2x80x9d encompasses fragments and analogues such as Fab, Fv and ScFv, provided that the binding activity is retained. Peptide fragments of antibodies are specifically contemplated by the invention. The fragments or analogues may be prepared using recombinant DNA methods or by synthetic methods such as solid-phase synthesis. The radioimaging may be conducted using Single Photon Emission Computer Tomography (SPECT), Position Emmission Tomography (PET), Computer Tomography (CT) or Magnetic Resonance Imaging (MRI). Correlative imaging, which permits greater anatomical definition of location of metastases located by radioimmunoimaging, is also contemplated.
In a fourth aspect, the invention provides a method of screening of therapeutic radiolabelled peptides directed against tumours, preferably tumour-associated receptors, antigens or ligands or the like. Therapeutic radiolabelled peptides such as 90 Yttrium-labelled octreotide or 111Indium-labelled octreotide are contemplated. Radiolabelled antibodies to tumour-associated ligands or antigens and therapeutic agents linked to such entities are also within the scope of the invention.
In a fifth aspect, the invention provides a method of producing a ruminant mammal bearing a tumour xenograft, comprising the step of concomitant administration of CsA and ketoconazole to the mammal. Preferably the ketoconazole is administered in a drench formulation which by-passes the rumen and is absorbed in the abomasum. This provides highly reproducible bioavalability and predictable competitive inhibition of CsA metabolism in the liver.
Preferably, the dose of CsA is in the range 2.5 to 3.5 mg per kg administered twice a day and the dose of ketoconazole is 5 to 10 mg per kg administered twice a day. In a particularly preferred embodiment, 10 mg/kg ketoconazole is administered twice a day to maintain trough serum levels of CsA within the range 1000-1500 ng/ml.
The model system of the invention enables the testing of therapeutic methods directed against primary malignancy or metastatic cancer in a manner which has hitherto not been possible. The model is suitable for testing of radiotherapy, immunotherapy (including the use of cytokines), chemotherapy, and gene therapy. The model is also useful for testing of targeting or localisation agents, methods of imaging, and methods for monitoring the progress of therapy.
In a sixth aspect, the invention provides a method of direct transplantation of a xenogeneic tumour, comprising the step of transplanting a surgically-removed specimen into a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole and allowing the specimen to metastasize in said mammal.
In a seventh aspect, the invention provides a method-of stimulating spontaneous metastasis of tumour cells to a target site such as the liver or lymph nodes, comprising the step of transplanting said cells to a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole and allowing the cells to metatasize in said mammal.
In a eighth aspect, the invention provides a composition comprising a CsA or cyclosporin-like compounds and ketoconazole or related compounds, together with a pharmaceutically acceptable carrier.
In a ninth aspect, the invention provides a kit comprising a CsA or cyclosporin-like compound and ketoconazole or a related compound, wherein the ketoconazole or related compound increases the bioavailability of the CsA or cyclosporin-like compound and enhances the establishment of tumour xenografts.
It will be clearly understood that, although the invention has been described in detail with reference to immunosuppression using CsA whose bioavailability is enhanced with ketoconazole, the invention also contemplates the use of immunosuppressive compounds related to CsA, such as those disclosed in U.S. Pat. No. 4,117,118, synthetic or natural analogues of CsA such as CsB to I, or the compounds disclosed in Australian Patent No. 660623 by Vertex Pharmaceuticals, Inc.
In addition, there are other compounds which the person skilled in the art will recognise as being suitable to improve bioavailability of CsA, such as compounds related to ketoconazole (including, but not limited to, fluconazole), and calcium channel blockers.
Without wishing to be bound by any proposed mechanism for the observed advantages, it is believed that ketoconazole, which bears no chemical structural relationship to CsA, competes with hepatic enzymes which break down CsA.